Project Summary: We propose to use genetically-engineered T cells to deliver cytotoxic cytokines under the control of a radio-inducible promoter. When the genetically engineered T cells localize to the tumor bed, activation of the cytokine is achieved by radiotherapy (RT), thereby exposing the tumor to the therapeutic effects of both RT and cytokine therapy. This ?molecular switch? strategy was pioneered by our laboratory, employing recombinant adenoviral vectors. In this concept, RT activates a chimeric gene with a radio-inducible promoter linked to a cytokine gene. Furthermore, the direct anti-tumor effects of RT have the potential to synergize with the anti-tumor effects of adoptively-transferred T cells. HYPOTHESIS: The ability to exogenously control the production of cytokines by tumor-infiltrating T cells will (i) counteract the immunosuppressive effects of the tumor, affecting a critical anti-tumor T cell effector function, (ii) minimize systemic toxicity due to controlled local activation of cytokine production, (iii) potentiate the synergy between RT and adoptive transferred T cells, and therefore (iv) improve the therapeutic ratio of RT. Specific Aims: 1) Cloning of T cell-specific radio-inducible promoters in retroviral vectors driving expression of a reporter gene for transduction of T cells, and in vivo analysis of gene induction by RT using longitudinal tumor imaging; 2) Cloning of T cell-specific radio-inducible promoters in retroviral vectors to drive expression of therapeutic cytokines in T cells and treatment of murine primary and oligometastatic tumors with radio-inducible cytokines-expressing T cells plus local RT. Our imaging technology uniquely allows us to examine the same tumor, and an identical area of the tumor, hours or days after each RT dose, and measure the increase in EGFP expression compared to levels before RT. This will allow in vivo comparison and validation of radio-induction of T cell-specific promoters identified in Aim 1. Furthermore, anti-tumor effects of T cells will be visible as destruction of cancer cells and vessels, thereby providing us with mechanistic data. Other cutting-edge technologies in our proposal include a CT-scan image- guided small animal irradiator for irradiation of mouse cancer tissues with millimetric precision. Our proposed combination of RT, gene therapy and adoptive T-cell therapy for treatment of locally advanced or metastatic cancer is dramatically new, and therefore has the potential to move beyond traditional approaches in the treatment of cancer. The initial clinical application of our new paradigm is the treatment of refractory solid tumors and oligometastases with the combined effects of adoptively-transferred T cells carrying radio-inducible cytokine cargo and RT, since RT has been shown to be an immunostimulatory modality; therefore, it is likely that translation of our approach would result in increased cures and significantly lower toxicities for cancer patients. Of note, our system could be applied to any other therapeutic gene that needs to be activated locally.